Abstract
From gene expression to nanotechnology, understanding and controlling DNA requires a detailed knowledge of its higher order structure and dynamics. Here we take advantage of the environment-sensitive photoisomerization of cyanine dyes to probe local and global changes in DNA structure. We report that a covalently attached Cy3 dye undergoes strong enhancement of fluorescence intensity and lifetime when stacked in a nick, gap or overhang region in duplex DNA. This is used to probe hybridization dynamics of a DNA hairpin down to the single-molecule level. We also show that varying the position of a single abasic site up to 20 base pairs away modulates the dye–DNA interaction, indicative of through-backbone allosteric interactions. The phenomenon of stacking-induced fluorescence increase (SIFI) should find widespread use in the study of the structure, dynamics and reactivity of nucleic acids.
Highlights
The environmental sensitivity of cyanine dyes such as Cy3 in the vicinity of a protein, known as protein-induced fluorescence enhancement (PIFE), is a powerful approach to studying DNA–protein interactions [1,2]
We took advantage of the dye’s sensitivity to local DNA structure, revealing long-range perturbations due to the presence of a single abasic site up to 20 base pairs away from the site of dye stacking; we discuss this finding in the context of the recent report of allostery in DNA–protein interactions [6]
We first designed a fully complementary DNA hairpin labelled with the cyanine dye Cy3 attached to the 3 end of the stem region (Figure 1A and Supplementary Figure S1) [8,10]
Summary
The environmental sensitivity of cyanine dyes such as Cy3 in the vicinity of a protein, known as protein-induced fluorescence enhancement (PIFE), is a powerful approach to studying DNA–protein interactions [1,2]. The more sterically hindered the rotation of the double bond is, the longer Cy3 spends in the trans conformation, resulting in an increased emission quantum yield [5]. This allows the dye to report on its microenvironment. We took advantage of the dye’s sensitivity to local DNA structure, revealing long-range perturbations due to the presence of a single abasic site up to 20 base pairs (bp) away from the site of dye stacking; we discuss this finding in the context of the recent report of allostery in DNA–protein interactions [6]
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